Virological and Serological Predictors of Anal High-grade Squamous Intraepithelial Lesions Among Human Immunodeficiency Virus-positive Men Who Have Sex With Men.

BACKGROUND: Our objective was to identify virological and serological predictors of anal high-grade squamous intraepithelial lesions (HSIL) in human immunodeficiency virus (HIV)-positive men who have sex with men (MSM). METHODS: HIV-positive MSM were recruited from a longitudinal study during which anal self-swabs and serum were collected at up to 5 bi-annual visits. Swabs were human papillomavirus (HPV) genotyped, and the type-specific HPV viral load in the anal swabs was determined. Serum antibodies to the E6, E7, E1, E2, and L1 proteins of 7 high-risk HPV (hrHPV) types and HPV6 and 11 were analyzed. The participants who had a high-resolution anoscopy after the last study visit were included in the current analysis. Anal HSIL was diagnosed by histopathological examinations of anal biopsies. The causative HPV type of anal HSIL was determined in whole tissue sections (WTS) and by laser capture micro-dissection if more than one HPV-type was found in WTS. Multivariable logistic regression was used to study whether persistent anal HPV infections, HPV viral loads, and seropositivity for HPV were predictors of anal HSIL, either in general or caused by the concordant HPV type. RESULTS: Of 193 HIV-positive MSM, 50 (26%) were diagnosed with anal HSIL. HrHPV persistence in anal swabs was common, varying by hrHPV type between 3-21%. Anal HPV persistence was the only determinant independently associated with anal HSIL, both in general and by concordant, causative HPV type. CONCLUSIONS: Persistent HPV infections were strongly associated with anal HSIL, in general as well as for the concordant HPV type.

Reliable identification of women with CIN3+ using hrHPV genotyping and methylation markers in a cytology-screened referral population.

Cervical screening aims to identify women with high-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia 2-3 (HSIL/CIN2-3) or invasive cervical cancer (ICC). Identification of women with severe premalignant lesions or ICC (CIN3+) could ensure their rapid treatment and prevent overtreatment. We investigated high-risk human papillomavirus (hrHPV) detection with genotyping and methylation of FAM19A4/miR124-2 for detection of CIN3+ in 538 women attending colposcopy for abnormal cytology. All women had an additional cytology with hrHPV testing (GP5+/6+-PCR-EIA+), genotyping (HPV16/18, HPV16/18/31/45), and methylation analysis (FAM19A4/miR124-2) and at least one biopsy. CIN3+ detection was studied overall and in women <30 (n = 171) and ≥30 years (n = 367). Positivity for both rather than just one methylation markers increased in CIN3, and all ICC was positive for both. Overall sensitivity and specificity for CIN3+ were, respectively, 90.3% (95%CI 81.3-95.2) and 31.8% (95%CI 27.7-36.1) for hrHPV, 77.8% (95%CI 66.9-85.8) and 69.3% (95%CI 65.0-73.3) for methylation biomarkers and 93.1% (95%CI 84.8-97.0) and 49.4% (95%CI 44.8-53.9) for combined HPV16/18 and/or methylation positivity. For CIN3, hrHPV was found in 90.9% (95%CI 81.6-95.8), methylation positivity in 75.8% (95%CI 64.2-84.5) and HPV16/18 and/or methylation positivity in 92.4% (95%CI 83.5-96.7). In women aged ≥30, the sensitivity of combined HPV16/18 and methylation was increased (98.2%, 95%CI 90.6-99.7) with a specificity of 46.3% (95%CI 40.8-51.9). Combination of HPV16/18 and methylation analysis was very sensitive and offered improved specificity for CIN3+, opening the possibility of rapid treatment for these women and follow-up for women with potentially regressive, less advanced, HSIL/CIN2 lesions.

HPV E4 expression and DNA hypermethylation of CADM1, MAL, and miR124-2 genes in cervical cancer and precursor lesions.

In this study, we evaluate the expression of human papillomavirus E4 protein (marker for the onset of a productive infection) and hypermethylation of host-cell CADM1, MAL, and miR124-2 genes (marker for an advanced, transforming infection) in cervical intraepithelial neoplasia (CIN) and cancer. A total of 115 cervical lesions were categorized by 3 pathologists into no dysplasia, CIN1, CIN2, CIN3, or cancer by classical histomorphological grading criteria, and by an immunoscore (cumulative value: 0-6) grading system based on Ki-67 (score: 0-3) and p16ink4a (score: 0-3) expression. Lesions were immunostained for E4 protein and analyzed for hypermethylation of CADM1, MAL, or miR124-2 genes. Expression of E4 and hypermethylation levels were related to CIN grade based on both classical and immunoscore grading. Hypermethylation increased with severity of the lesion as defined by both classical histomorphological grading and immunoscore criteria, and was always present in carcinomas (22/22). Extensive E4 expression decreased with increasing CIN grade and immunoscore, being most frequent in classically graded CIN1 or in lesions with cumulative immunoscore 1-3 and absent in carcinomas. High-grade lesions (CIN2/3 or immunoscore: 4-6) showed less E4 expression, which was inversely related to an increasing hypermethylation. Extensive E4 expression, as observed in a small proportion of high-grade lesions (6/49 and 8/43, respectively), was mostly associated with a negative methylation marker status (5/6 and 7/8, respectively). Our results illustrate the gradual transition of productive CIN (reflected by extensive E4 expression), to advanced transforming CIN (reflected by extensive hypermethylation) and cancer. Expression patterns of E4 and hypermethylation status of host-cell genes, may be used to identify cervical lesions at risk for cervical cancer, providing a better guidance for clinicians on treatment decisions.

Defining hrHPV genotypes in cervical intraepithelial neoplasia by laser capture microdissection supports reflex triage of self-samples using HPV16/18 and FAM19A4/miR124-2 methylation.

OBJECTIVE: HPV16/18 genotyping and detection of hypermethylation of human cell genes involved in cervical oncogenesis have shown promising results in triage of high-risk HPV (hrHPV)-screen positive women on cervical smears. These tests can be performed on self-samples, which contain cervical and vaginal cells. We studied whether a self-sample represents the hrHPV type causing the worst cervical lesion and whether any differences in hypermethylation of FAM19A4/miR124-2 exist between CIN lesions caused by different hrHPV types. These results have important implications for reflex triage of self-samples. METHODS: Correlation between genotype found on self-sample using GP5+/6+-PCR-EIA-LMNX and causative hrHPV genotype in the worst lesion on histology was studied using laser capture microdissection (LCM)-SPF10-PCR (N = 152). Hypermethylation of FAM19A4/miR124-2 in the self-sample was tested in a quantitative methylation specific PCR and compared between lesions caused by HPV16/18 and other hrHPV genotypes. RESULTS: Causative hrHPV genotype of the worst lesion (CIN1, CIN2, CIN3, invasive cervical cancer) was detected on self-sample in 93.4%. HPV16 was the most frequently found genotype on self-sampling (39.2%, 73/186) and causative genotype in CIN3+ (51.4%, 38/74, all detected on self-sample). There were no differences in the percentages of positive FAM19A4/miR124-2 methylation assays between lesions caused by HPV16/18 (73.8% in CIN3+) or other hrHPV genotypes (66.7% in CIN3+) (p = 0.538). CONCLUSIONS: Our results show that hrHPV genotypes found on self-sample were a good representation of hrHPV in the worst CIN lesion and that methylation testing on self-sample for detection of CIN3+ was not significantly different between lesions caused by HPV16/18 and other hrHPV genotypes.

A Low-Grade Trichoblastic Carcinoma Treated with Mohs Micrographic Surgery.

Trichoblastomas are rare dermal neoplasms usually found on the scalp and face. Histology shows a proliferation of small basaloid cells arranged in cords or fields surrounded by cellular stroma. Trichoblastomas are usually not aggressive, but trichoblastic carcinomas arising from preexisting trichoblastomas have been described and have been linked to basal cell carcinoma. We found a tumor with features of trichoblastoma with presence of Merkel cells, but with a deeply infiltrative growth pattern into the fat and muscle tissue, without significant architectural or cellular atypia. Tumors with similar growth patterns were previously described as deeply invasive trichoblastic neoplasms. It appears to be a new entity that has been described before but has not been fully characterized: low-grade trichoblastic carcinoma. This malignancy seems to show only locally aggressive growth. Radical excision was accomplished with Mohs micrographic surgery.

Expression of p16 and HPV E4 on biopsy samples and methylation of FAM19A4 and miR124-2 on cervical cytology samples in the classification of cervical squamous intraepithelial lesions.

The decision to treat a cervical squamous intraepithelial lesion (SIL) by loop electrosurgical excision procedure (LEEP) relies heavily on a colposcopy-directed biopsy showing high-grade (H)SIL. Diagnosis is often supported by p16, an immunohistochemical (IHC) biomarker of high-risk (hr)HPV E7 gene activity. Additional potential markers include methylation of tumor suppressor genes FAM19A4/miR124-2 in cervical cytology for advanced transforming HSIL and the IHC marker HPV E4 for productive, potentially regressing lesions. In 318 women referred for colposcopy, we investigated the relationship between staining patterns of p16 and E4 IHC in the worst biopsy, and the relation of these to FAM19A4/miR124-2 methylation status in cytology. E4-positive staining decreased with increasing SIL/CIN grade from 41% in LSIL to 3% in HSIL/CIN3. E4 positivity increased with grade of p16 when p16 expression was limited to the lower two third of the epithelium (r = 0.378), but fell with expression over. Loss of E4 expression in the worst lesion was associated with the methylation of FAM19A4/miR124-2. We also examined whether these biomarkers can predict the histological outcome of the LEEP biopsy in a subgroup of 119 who underwent LEEP treatment. About 85% of women with ≥lower two third p16 staining/E4-negative HSIL biopsies and 65% with limited p16 staining/E4-positive HSIL biopsies had ≥HSIL in the LEEP specimen (P = .025). p16 expression in a biopsy is related both to viral production and transformation, while decreased E4 expression relates to methylation, indicating advanced HSIL. p16 expression in ≥2/3 of the epithelium and absent E4 indicate likely HSIL on a subsequent LEEP specimen.

Utility of Stathmin-1 as a Novel Marker in Evaluating Anal Intraepithelial Neoplasia (AIN).

The aim of this study is to determine whether immunohistochemistry for Stathmin-1 enhances diagnostic accuracy of anal dysplasia. The study included 40 biopsies with diagnosis of benign anal transitional zone (n=10), low-grade anal intraepithelial neoplasia (AIN) (AIN1, n=10), and high-grade AIN (AIN2, n=10, AIN3, n=10). The cases were selected to represent classic features. Immunohistochemistry for Stathmin-1, p16, and Ki-67 was performed and assessed for distribution within epithelial thickness. Stathmin-1 was expressed only in the basal layer of benign anal epithelium. Similar pattern of distribution was seen in all low-grade AIN cases (100%). In total, 40% of AIN2 showed Stathmin-1 staining pattern similar to AIN1. The other 60% of cases showed staining extending into the middle third of the epithelial thickness. Of AIN3 cases, 20% showed staining confined to the lower third epithelium, 20% showed staining extending to the middle third, and 60% showed staining extending into the upper third epithelium. The pattern of stain distribution suggested that staining extending above the lower one-third of the epithelial thickness discriminates between low-grade and high-grade AIN. With this cutoff, the sensitivity for the diagnosis of high-grade AIN was 70%, specificity was 100%, positive predictive value equaled 100%, and negative predictive value equaled 77%. P16INK4a showed 100% sensitivity for AIN2 and AIN3, whereas Ki-67 had 100% sensitivity for any AIN grade. In conclusion, Stathmin-1 has excellent specificity for the diagnosis of high-grade AIN; however, Stathmin-1 alone may not be sufficiently sensitive. Use in conjunction with other sensitive markers, such as p16 or Ki-67 may be considered.

Presence or Absence of Significant HPVE4 Expression in High-grade Anal Intraepithelial Neoplasia With p16/Ki-67 Positivity Indicates Distinct Patterns of Neoplasia: A Study Combining Immunohistochemistry and Laser Capture Microdissection PCR.

Progression of anal intraepithelial neoplasia (AIN) involves transition from productive to transforming human papillomavirus (HPV) infection. Grading aims to distinguish productive low-grade AIN from high-grade anal intraepithelial neoplasia (HGAIN) with risk of cancer. We describe immunohistochemical patterns in AIN adding a novel marker for initiation of the productive phase of the HPV life cycle (panHPVE4) to those for cell cycle activity (Ki-67) and transforming activity of HPVE7 gene (p16). We studied 67 anal biopsies for suspected anal neoplasia (17 normal, 15 AIN1, 20 AIN2, 15 AIN3) from 54 men who have sex with men at New York Presbyterian Hospital, USA. Two pathologists generated consensus AIN and immunogrades. Whole tissue and laser capture microdissection samples from multiple HPV-infected biopsies were tested for HPV with SPF10-PCR-DEIA-LiPA25, version 1. (Para)basal Ki-67 expression distinguished normal from AIN (≥lower-third Ki-67) with sensitivity 0.92 and specificity 1.0. Ki-67 did not distinguish grades of AIN. Null/patchy p16 versus diffuse ≥lower-third patterns discriminated HGAIN (sensitivity, 1.0; specificity, 0.84). There was marked heterogeneity in E4 expression within HGAIN. Most AIN2 (14/20) was E4 versus 0/15 AIN3 (sensitivity, 0.70; specificity 1.0). HPV was detected in 63 (94%) biopsies, with 49 (77.8%) high-risk HPV. HPV16 was the most frequent (13%). Multiple HPV genotypes were found in 15 (24%) biopsies and laser capture microdissection -polymerase chain reaction confirmed specific HPV types in E4 +/- AIN. Although Ki-67 discriminated AIN and p16 HGAIN, E4/p16 staining shows that most AIN2 is different from transformed AIN3 in showing both entry into productive HPV infection and transforming activity.

Three-tiered score for Ki-67 and p16ink4a improves accuracy and reproducibility of grading CIN lesions.

AIMS: To investigate the accuracy and reproducibility of a scoring system for cervical intraepithelial neoplasia (CIN1-3) based on immunohistochemical (IHC) biomarkers Ki-67 and p16ink4a. METHODS: 115 cervical tissue specimens were reviewed by three expert gynaecopathologists and graded according to three strategies: (1) CIN grade based on H&E staining only; (2) immunoscore based on the cumulative score of Ki-67 and p16ink4a only (0-6); and (3) CIN grade based on H&E supported by non-objectified IHC 2 weeks after scoring 1 and 2. The majority consensus diagnosis of the CIN grade based on H&E supported by IHC was used as the Reference Standard. The proportion of test positives (accuracy) and the absolute agreements across pathologists (reproducibility) of the three grading strategies within each Reference Standard category were calculated. RESULTS: We found that immunoscoring with positivity definition 6 yielded the highest proportion of test positives for Reference Standard CIN3 (95.5%), in combination with the lowest proportion of test positives in samples with CIN1 (1.8%). The proportion of test positives for CIN3 was significantly lower for sole H&E staining (81.8%) or combined H&E and IHC grading (84.8%) with positivity definition ≥CIN3. Immunoscore 6 also yielded high absolute agreements for CIN3 and CIN1, but the absolute agreement was low for CIN2. CONCLUSIONS: The higher accuracy and reproducibility of the immunoscore opens the possibility of a more standardised and reproducible definition of CIN grade than conventional pathology practice, allowing a more accurate comparison of CIN-based management strategies and evaluation of new biomarkers to improve the understanding of progression of precancer from human papillomavirus infection to cancer.

Randomized trial evaluating self-sampling for HPV DNA based tests for cervical cancer screening in Nigeria.

BACKGROUND: Cervical cancer incidence and mortality rates in Sub-Saharan Africa (SSA) remain high due to several factors including low levels of uptake of cervical cancer screening. Self-collection of cervicovaginal samples for HPV DNA testing may be an effective modality that can increase uptake of cervical cancer screening in SSA and hard to reach populations in developed countries. We investigated whether self-collection of cervicovaginal samples for HPV DNA tests would be associated with increased uptake of screening compared with clinic based collection of samples. Furthermore, we compared the quality of samples collected by both approaches for use in HPV genotyping. METHODS: We conducted a community based randomized trial in a semi-urban district of Abuja, Nigeria with 400 women, aged 30 to 65 years randomized to either hospital-collection or self-collection of cervicovaginal samples. We compared cervical cancer screening uptake among the 2 groups and evaluated the concentration of human DNA in the samples by measuring RNase P gene levels using qPCR. High-risk HPV DNA detection and typing was done using the GP5+/6+ Luminex system. RESULTS: Most participants in the self-collection arm (93%, 185/200) submitted their samples while only 56% (113/200) of those invited to the hospital for sample collection attended and were screened during the study period (p value < 0.001). Human genomic DNA was detected in all but five (1.7%) participants, all of whom were in the self-collection arm. The prevalence of high-risk HPV in the study population was 10% with types 35, 52 and 18 being the commonest. CONCLUSIONS: Our study shows that self-sampling significantly increased uptake of HPV DNA based test for cervical cancer screening in this population and the samples collected were adequate for HPV detection and genotyping. Cervical cancer screening programs that incorporate self-sampling and HPV DNA tests are feasible and may significantly improve uptake of cervical cancer screening in SSA.

Assessment and care for non-medical risk factors in current antenatal health care.

OBJECTIVE: this study aims to identify current practice in risk assessment, current antenatal policy and referral possibilities for non-medical risk factors (lifestyle and social risk factors), and to explore the satisfaction among obstetric caregivers in their collaboration with non-obstetrical caregivers. DESIGN: cross-sectional study SETTING: Dutch antenatal care system PARTICIPANTS: community midwives from 139 midwifery practices and gynaecologists, hospital-based midwives, and trainees in obstetrics from 38 hospitals. MEASUREMENTS AND FINDINGS: results were analysed with χ(2) tests and unpaired t-tests. Caregivers universally screened upon lifestyle risk factors (e.g. smoking or drug use), whereas the screening for social risk factors (e.g. social support) was highly variable. As national guidelines are absent, local protocols were reported to be used for screening on non-medical risk factors in more than 40%. Caregivers stated multidisciplinary protocols to be a prerequisite for assessment of non-medical risk factors. Only 22% of the caregivers used predefined criteria to define when patients should be discussed multidisciplinary. CONCLUSION: despite their relevance, non-medical risk factors remain an underexposed topic in antenatal risk factor screening in both the community and hospital-based care setting. Implications for practice Structural antenatal risk assessment for non-medical risk factors with subsequent consultation opportunities is advocated, preferably based on a multidisciplinary guideline.